Oocyte-derived R-spondin2 as a Follicle Stimulating Hormone Abstract: The Wingless (Wnt) signaling pathway is essential for cell proliferation from flies to mammals. Although multiple Wnt ligands and their cognate Frizzled receptors are expressed in ovarian somatic cells, there is no report demonstrating the ability of Wnt ligands in the promotion of ovarian follicle growth. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of primary and more advanced follicles but not in oocytes of dormant primordial follicles. There are four R-spondin paralogous genes (R-spondin1-4) in the human genome and these secreted proteins have been found to be potent growth factors for adult stem cells in gastrointestinal organs and hair follicles by serving as co-ligands for the Wnt signaling pathway essential for cell proliferation. Recent studies further demonstrated the role of LGR4/5/6 proteins as the cognate receptors for R-spondin ligands. Our preliminary data demonstrated the ability of R-spondin2 to synergize with Wnt ligands in the activation of the Wnt signaling pathway in cultured ovarian somatic cells. R-spondin2 treatment also promoted ovarian follicle development in cultured ovarian explants. Unlike FSH, R-spondin2 did not activate the cAMP-protein kinase A pathway and showed additive stimulation of ovarian cell proliferation together with FSH. We further generated a secreted R- spondin agonist, R-spondin1-Fc, by fusing R-spondin1 cDNA with that for the Fc domain of IgG. We showed the ability of R-spondin1-Fc to promote the growth of primary follicles to the secondary stage in neonatal mice in vivo. Pre-treatment with R-spondin1-Fc led to the induction of early antral follicles capable of responding to sequential eCG and hCG treatment, leading to the generation of mature oocytes. These oocytes could be fertilized in vitro and developed into blastocysts. Although FSH has been used extensively for the treatment of female infertility, a sub-population of patients showed low FSH responses and has no alternative therapeutic options. The present R21 proposal will first demonstrate the ovarian expression of R-spondin2 ligand and LGR4/5/6 receptor proteins, together with the mediatory role of ovarian LGR4/5/6 receptors. This will be followed by the generation of recombinant R-spondin2 to stimulate early follicle growth in neonatal wild type mice. Following the promotion of early ovarian follicle development using R-spondin2, mice will be treated sequentially with eCG and hCG to stimulate the final phase of follicle development and to derive mature oocytes for fertilization and the derivation of pups. We will then use a rodent model of low FSH responsiveness (FSH receptor haploinsufficient heterozygous mutants) to test the ability of R-spondin2 in the promotion of early follicle development for subsequent gonadotropin stimulation and the generation of mature oocytes. We will evaluate epigenetic changes, mitochondrial integrity and early embryonic development of mature oocytes to insure the efficacy and safety of the present treatment regimen. This will be followed by the derivation of healthy pups as the basis for future clinical application using R-spondin2 to treat infertile patients with low FSH responses. PUBLIC HEALTH RELEVANCE: The present application represents a new approach for female infertility treatment by demonstrating the ability of oocyte-derived R-sponind2 to promote early ovarian follicle development using wild type and FSH receptor haploinsufficient mice. The generation and use of recombinant R-spondin2 molecules could provide a new therapeutic agent for the treatment of infertile patients with low FSH responses.